Loop-mediated Isothermal Amplification (LAMP) as a diagnostic tool in detection of infectious diseases Dorota DRAPAŁA, Milena KORDALEWSKA Keywords: LAMP; DNA amplification; isothermal reaction Abstract: Loop-mediated isothermal amplification (LAMP) is a gene amplification method which amplifies DNA with high specificity and efficiency under isothermal conditions. Because of its rapidity and simplicity, it is a valuable diagnostic tool in the early detection and identification of infectious diseases. LAMP method is based on the use of a set of four to six specially designed primers spanning six to eight distinct sequences on the target DNA. The amplification is performed by DNA polymerase, which has strand-displacing activity. The whole reaction occurs in a single tube and is divided into two steps: non-cyclic and cyclic. The final products are artificial stem-loop DNA structures flanking the target sequences. 1. Introduction At present, techniques based on the detection of nucleic acids are used more and more often in the routine diagnostics of infectious diseases. PCR methods such as nested PCR, multiplex PCR, reverse transcription PCR or real-time PCR are commonly applied due to their sensitivity, specificity and rapidity (Van Belkum and Niesters, 1995). However, in spite of their many advantages, all the above-mentioned techniques have several significant limitations such as expensive amplification instruments, complicated methods of detection or complex analysis of the results. Loop-mediated isothermal amplification (LAMP) appeared to be an innovative technique which may be an alternative method for the diagnosis of some infectious diseases. It is sensitive, specific, fast (without time-consuming analysis) and, what is really significant, does not require expensive instruments and complicated methods of detection. 2. LAMP description LAMP was developed in 1998 by Eiken Chemical Company (Japan). It is a quite complex method which requires four to six different primers that are designed specifically Microbiology Department, Faculty of Chemistry, Gdańsk University of Technology, Narutowicza St. 11/12,80-233 Gdańsk
20 PhD Interdisciplinary Journal to recognize six to eight specific gene sequences. DNA amplification is accomplished with the use of DNA polymerase with strand-displacing activity (Notomi et al., 2000; Ushikubo, 2004). Thanks to this, there is no need for a thermal denaturation step in order to obtain single-stranded DNA. Thus, strand-displacing activity allows amplification under isothermal conditions in contrast to PCR where a thermal denaturation step is essential. LAMP technique is based on a generation of artificial stem-loop DNA structures flanking the target sequences. Cyclic strand displacement is performed at constant temperature, ca 65 C, at which double-stranded DNA remains in the dynamic equilibrium. This allows primers to anneal to the complementary sequence of DNA, so DNA polymerase (with strand-displacing activity) can start DNA synthesis. The whole LAMP procedure consists of two steps: non-cyclic and cyclic (Fig.1). The first step leads to the generation of artificial stem-loops used in the further steps (Fig. 1a). Firstly, the forward inner primer (FIP Primer) binds to the target sequence and initiates the polymerization. After that, the forward outer primer (F3 Primer) binds to the product and displaces it with a single artificial stem-loop appending to the target sequence. The single strand DNA serves as a temple for BIPinitiated (BIP Primer) DNA synthesis. Then, the reverse outer primer (B3 Primer) binds to the product and displaces the product with two artificial stem loops flanking the target reagent. This is the starting structure for the next, cyclic amplification step. The second cyclic step starts with the FIP primer hybridization to the loop on the product (Fig. 1b). Displacement DNA synthesis is initiated and leads to the generation of DNA with a new stem-loop structure at one end and an additional target sequence. These structures are used as templates in further DNA synthesis with the use of internal primers. Various-sized structures possessing the alternately inverted repeats of the target sequence on the same strand are formed. 3. LAMP advantages and disadvantages LAMP method, due to its sensitivity, specificity, high efficiency, rapidity and simplicity is a good molecular technique for identifying some infectious diseases. The main advantage of LAMP is the fact that amplification is performed under isothermal conditions. Therefore, it is possible to obtain high amplification efficiency, which is attributed to reduced time loss of thermal change. Moreover, there is no need for expensive and complicated equipment such as a thermal cycler. The process may be performed simply in a heating block or water bath. High specificity is obtained because of the use of 4-6 primers spanning 6-8 distinct sequences and all target genes must be present in order to initiate amplification. Additionally, the method is rapid and simple; amplification and detection may be carried out in one single tube. Gene amplification products can be detected not only by agarose gel electrophoresis and real-time monitoring in an inexpensive turbidimeter but also visually by the naked eye, either as turbidity or in the form of a colour change. In spite of its many advantages, LAMP also has some limitations. The primer designing is quite complicated, because it is essential to design 4-6 specific primers. Moreover, it must be remembered that LAMP is inadequate for the detection of unknown or unsequenced targets.
Special Issue: Biotech Conference 21 Tab. 1. LAMP applications in diagnostics Pathogens Disease Sequence for specific primers Method of detection References coronavirus SARS 1bRep gene spectrophotometric analysis by recording the optical density with a real-time turbidimeter (Hong et al., 2004) Mycobacterium tuberculosis,m. avium, M. intracellulare mycobacteriosis gyrb gene visualization by addition of SYBR Green I to the reaction tube (Iwamoto et al., 2003) Plasmodium falciparum malaria 18S ribosomal RNA gene analysis by turbidimeter in real-time or visually at the end of the assay (Poon, 2006) Staphylococcus aureus sepsis 16S ribosomal RNA gene analysis by agarose gel electrophoresis and visually by distinguished precipitation or turbidity (Zhang, 2013) Human immunodeficiency virus-1 HIV highly conserved sequences located within the protease and p24 gene regions identification by agarose gel electrophoresis and visually following the addition of the fluorescent nucleic acid stain (Curtis et al., 2008, 2009, 2012) Streptococcus pneumoniae pneumonia lyta gene analysis by agarose gel electrophoresis and visually by distinguished precipitation or turbidity (Seki et al., 2005)
22 PhD Interdisciplinary Journal Fig. 1. Non-cyclic (A) and cyclic (B) steps of LAMP Parida et al. (2008).
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